In cells, a majority of unwanted and harmful proteins are eliminated through a process called the ubiquitin-proteasome pathway. In this process, a target protein is tagged with a chain of ubiquitin molecules and the resulting marked protein is then recognized by the proteasome, which drives protein degradation. Proper functioning of the ubiquitin tagging and proteasomal activities is of key importance for the function of a cell and an organism. Defective regulation of the ubiquitin-proteasome system is manifest in human diseases including cancers. Our long-range goal is to understand the precise functioning of the SCF E3 ubiquitin ligase complex, which is the key machinery in cells responsible for tagging a large set of proteins with a chain of ubiquitin for degradation. The short-term objective of our project is to determine how a chain of ubiquitin is built on a protein, which signals degradation. A protein target has a built-in "degradation code" that dictates its destruction. In an ubiquitin molecule, "conjugation codes" are present to direct the formation of an ubiquitin chain, or a linkage that connects an ubiquitin to the protein target. The ubiquitin tagging reaction can be viewed as a process, beginning with the "reading" of the "degradation code" on the protein target, and ubiquitin "conjugation codes," by protein facilitators that include SCF E3 and enzymes such as UbcH5 and Cdc34. Subsequently, SCF, UbcH5, and Cdc34 act to "build" a large protein network that links the protein target with a chain of ubiquitin. However, it remains poorly understood regarding the nature of the ubiquitin "conjugation codes," how these codes are "read," as well as the network "building" process necessary for ubiquitin tagging. Using the tools of biochemistry, molecular biology and cell biology, our project is to: 1) identify new molecular components that help form the complex of a protein target and an SCF E3, which triggers the ubiquitin tagging process;2) understand how SCF, UbcH5, and Cdc34 build protein networks for tagging a target protein with ubiquitin;3) identify the ubiquitin "conjugation codes" and uncover the mystery of how Cdc34 reads these "codes." PUBLIC HEALTH RELEVANCE: This project intends to uncover the mystery of tagging a protein target with a chain of ubiquitin molecules, which leads to protein degradation. Knowledge gained in this study will advance understanding of this ubiquitin tagging process that is fundamental to the function of a cell and an organism. Importantly, the molecular insights revealed in this project will improve the designing of drugs effective for treating human diseases that include cancer.